The impact of mosquito sampling strategies on molecular xenomonitoring prevalence for filariasis: a systematic review

Abstract Objective To explore the impact of mosquito collection methods, sampling intensity and target genus on molecular xenomonitoring detection of parasites causing lymphatic filariasis. Methods We systematically searched five databases for studies that used two or more collection strategies for sampling wild mosquitoes, and employed molecular methods to assess the molecular xenomonitoring prevalence of parasites responsible for lymphatic filariasis. We performed generic inverse variance meta-analyses and explored sources of heterogeneity using subgroup analyses. We assessed methodological quality and certainty of evidence. Findings We identified 25 eligible studies, with 172 083 mosquitoes analysed. We observed significantly higher molecular xenomonitoring prevalence with collection methods that target bloodfed mosquitoes compared to methods that target unfed mosquitoes (prevalence ratio: 3.53; 95% confidence interval, CI: 1.52–8.24), but no significant difference compared with gravid collection methods (prevalence ratio: 1.54; 95% CI: 0.46–5.16). Regarding genus, we observed significantly higher molecular xenomonitoring prevalence for anopheline mosquitoes compared to culicine mosquitoes in areas where Anopheles species are the primary vector (prevalence ratio: 6.91; 95% CI: 1.73–27.52). One study provided evidence that reducing the number of sampling sites did not significantly affect molecular xenomonitoring prevalence. Evidence of differences in molecular xenomonitoring prevalence between sampling strategies was considered to be of low certainty, due partly to inherent limitations of observational studies that were not explicitly designed for these comparisons. Conclusion The choice of sampling strategy can significantly affect molecular xenomonitoring results. Further research is needed to inform the optimum strategy in light of logistical constraints and epidemiological contexts.


Introduction
Lymphatic filariasis is a disabling and debilitating disease caused by the filarial worms Wuchereria bancrofti, Brugia malayi or B. timori that are transmitted by mosquitoes of the genera Culex, Anopheles, Aedes and Mansonia.The disease is targeted for elimination using mass drug administration and vector control; many countries have already achieved the goal of elimination as a public health problem.The World Health Organization (WHO) recommends that countries continue disease surveillance using cross-sectional surveys, routine surveillance of target populations, or molecular xenomonitoring to ensure infection levels remain below target thresholds or to confirm interruption of transmission. 1olecular xenomonitoring is used as a surveillance strategy for vector-borne diseases such as lymphatic filariasis and onchocerciasis.The technique detects the presence of pathogen genetic material such as deoxyribonucleic acid (DNA) in disease vectors (e.g.mosquitoes).This method therefore gives a measure of the vector population's exposure to pathogens picked up from infected humans, allowing it to serve as a proxy for the presence of human disease.
Molecular xenomonitoring overcomes many of the key challenges of lymphatic filariasis case surveillance: it does not rely on human blood sampling, it is relatively inexpensive and it allows integrated surveillance of multiple diseases. 2Innovations in mosquito trap design and field-friendly amplification and detection techniques are now bringing molecular xenomonitoring into the reach of control programmes, even those that lack specialist entomology training. 3A metaanalysis showed that molecular xenomonitoring had a high sensitivity at low microfilaria prevalence in communities, and demonstrated a strong correlation between molecular xenomonitoring and microfilaria prevalence when a consistent method is applied. 4 major limitation of molecular xenomonitoring is that there is no standardized protocol for sampling mosquitoes. 3urrent WHO guidelines on molecular xenomonitoring 5 indicate that any collection method can be used, and provide minimal instruction on frequency, scale, target species and sample sizes.However, each of these variables may influence the likelihood of a molecular xenomonitoring survey detecting mosquitoes positive for filarial DNA.Consequently, standardized guidelines, along with those tailored to specific settings, are needed.These guidelines will ensure that collection strategies effectively detect areas of potential disease transmission, and that results are comparable across timepoints and evaluation units.Developing such guidelines for sampling mosquitoes requires an understanding of how collection strategies influence the prevalence of filarial DNA in wild-caught mosquitoes.
Several mosquito collection methods can be used for lymphatic filariasis xenomonitoring. 5Each method exploits a specific stage of the mosquito's gonotrophic cycle and therefore predominantly collects, though not exclusively, mosquitoes from that stage.In this review we have broadly categorized these methods into three groups: (i) fed collection methods, which target mosquitoes that have recently fed on blood.These methods use indoor resting catches (in which mosquitoes are collected either by direct aspiration or through the use of insecticides), or exit traps (traps fixed to windows to collect mosquitoes as they attempt to leave a building); (ii) gravid collection methods, which lure and trap gravid, ovipositing females using gravid traps; and (iii) unfed collection methods, which trap mosquitoes by exploiting their host-seeking behaviour.These methods use light traps (with or without carbon dioxide), odour-baited traps (such as the Biogents' BG-Sentinel trap, Regensburg, Germany) or human landing catches (in which mosquitoes are caught as they alight on a human collector).
As the presence of parasite DNA in the mosquito is dependent on a previous bloodmeal from a lymphatic filariasis-infected host, bloodfed or gravid mosquitoes have a higher likelihood of containing filarial DNA than unfed mosquitoes.
A second factor that may affect the molecular xenomonitoring prevalence is the intensity of the sampling protocol.Lymphatic filariasis is a highly focal disease, 6 and local transmission is influenced by a combination of environmental, climatic and socioeconomic conditions.Small hotspots of high transmission can persist in districts that have very low transmission overall. 7,8he likelihood of detecting a positive mosquito may increase with a higher number of sampling locations.
A third factor affecting molecular xenomonitoring prevalence is the predominant genus of mosquitoes collected.Molecular xenomonitoring prevalence is likely to be higher for mosquitoes that act as vectors for the disease than those that do not.In competent vectors, microfilariae that have been ingested develop into infective stage larvae, a process that takes a minimum of 10-12 days, so parasite DNA may be detectable for the remainder of the mosquito's life. 9,10In non-competent vectors, parasite DNA is transient and often expelled within 48 hours of bloodmeal ingestion. 11Another consideration is bloodmeal size; Culex quinquefasciatus can ingest twice as many microfilariae as Aedes aegypti under experimental conditions. 12Anthropophilic (humanseeking) vectors, such as Anopheles gambiae, are also likely to have greater exposure to filarial DNA than those that feed from a variety of hosts.
In this systematic review we aimed to determine how sampling strategy affects molecular xenomonitoring prevalence and informs molecular xenomonitoring implementation.We compared molecular xenomonitoring prevalence when measured using two or more different methods from within the following three categories: (i) mosquito collection methods: fed, gravid and unfed; (ii) sampling intensity; and (iii) mosquito genera: Anopheles, Culex, Mansonia, Aedes and Armigeres.

Methods
We followed the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines 13 using predetermined methods from a protocol registered with the PROSPERO international database of prospectively registered systematic reviews (registration number CRD42020200351). 14

Eligibility criteria
Studies were included in our review if they met all of the following criteria: (i) they used two or more collection strategies; (ii) wild mosquito populations were sampled; and (iii) they used molecular methods (polymerase chain reaction (PCR), genetic sequencing or loop-mediated isothermal amplification) to both measure and report the molecular xenomonitoring prevalence for the causative agents of lymphatic filariasis (W.bancrofti, B.malayi, B. timori).We did not apply any language restrictions to the inclusion criteria.

Search
We searched five bibliographic databases (CINAHL Complete, eBook Collection, Global Health, Global Health Archive and MEDLINE Complete) for all records up to and including 8 February 2023 using the EBSCOhost research platform. 15e also checked the reference lists in included studies to identify further studies meeting the inclusion criteria.The search strategy is outlined in Box 1. Two reviewers assessed abstracts and selected papers for full-text screening.
We resolved discrepancies by thorough review and discussion.

Data extraction
We extracted study information at the smallest available level, e.g.individual villages within a district.Where molecular xenomonitoring prevalence and associated 95% confidence intervals (CIs) were not reported, we calculated these using data reported in the study.Where mosquitoes were screened in pools, we estimated the molecular xenomonitoring prevalence and 95% CI from the total number of mosquitoes screened, the size of the pools and the proportion of pools that were positive using the Poolscreen algorithm, version 2.0. 17If the sizes of individual pools were not reported, we used the mean pool size for Poolscreen calculations.

Methodological quality assessment
We used the QUADAS-2 tool to assess the methodological quality of the included studies. 18The tool (available in the online repository) 19 was adapted for evaluation of community-level surveillance methods before the start of the screening process.

Statistical analysis and data synthesis
We calculated prevalence ratios with 95% CIs using RevMan 5 (Cochrane, London, United Kingdom of Great Britain and Northern Ireland) to compare molecular xenomonitoring prevalence between different sampling strategies.Because pooled mosquito data have a higher level of uncertainty than the same number of samples screened individually, we transformed the reported samples sizes to effective samples sizes (available in the online repository) 19 to ensure Rev-Man calculated the correct CIs.
We conducted all meta-analyses using generic inverse variance models  20 We performed subgroup analyses by filarial parasite species, primary vector genus and trapping methods used to explore reasons for substantial heterogeneity.To assess significant differences between subgroups, we used χ 2 -squared tests with P-values less than 0.1 deemed statistically significant.We also performed sensitivity analyses to evaluate the effect of exclusion of trials that had a high risk of bias for any of the QUADAS-2 domains.Where 10 or more studies were included in a meta-analysis, we investigated the risk of publication bias using funnel plots. 21e assessed the certainty that the true differences between sampling methods lie close to those estimated by our meta-analyses using the Grading of Recommendations Assessment, Development and Evaluation approach. 22s all the included studies were observational studies, the evidence for each outcome started as low certainty; this grade could be further downgraded due to concerns about any of the following five domains: risk of bias; imprecision; inconsistency; indirectness; and reporting bias. 23

Results
We identified 1142 records through electronic database searching, and three records through contact with study authors.After removal of duplicates, we screened 407 records.Of these, we identified 107 records for full-text assessment and 25 of these met the inclusion criteria. 3, Thes were included in qualitative and quantitative synthesis (Fig. 1).

Characteristics of included studies
Of the included studies, 12 studies were conducted in the WHO African Region, eight in the South-East Asia Region, two each in the Western Pacific Region and in the Region of the Americas, and one in the Eastern Mediterranean Region.The study characteristics are summarized in Table 1.A total of 172 083 mosquitoes were analysed across the included studies with a mean of 6373 per study (range: 208-57 357).

Methodological quality
We provide a summary of the methodological quality in Table 2.The sampling methods used in two studies were judged to introduce a high risk of bias. 26,39One study 26 screened mosquitoes caught by the indoor resting catch method in pools of 10, and those caught by human land-ing catch method in pools of 50; their study also reported reduced sensitivity of molecular detection in pool sizes of 50 compared to microscopic detection following dissection.This approach prevents an unbiased comparison of mosquitoes collected by the two catch methods.Another study 39 used light traps to preferentially collect Anopheles mosquitoes and gravid traps to preferentially collect Culex mosquitoes, and they presented their data according to species.The comparisons we have made between species are likely confounded by the fact that gravid traps collect mosquitoes that have taken at least one bloodmeal.

Summary of findings
Molecular xenomonitoring prevalence was significantly higher when fed collection methods were used compared with unfed.Higher prevalence was also observed in anopheline mosquitoes than culicine mosquitoes.However, the certainty of the evidence scored very low (Table 3).A summary of the assessments and the justifications for each rating are also provided in Table 3. Below, we discuss in detail the data underpinning each of these assessments.

Fed versus gravid
0][31] Higher molecular xenomonitoring prevalence was observed for mosquitoes collected using fed traps than for mosquitoes collected using gravid traps, but this difference was not statistically significant (prevalence ratio: 1.54; 95% CI: 0.46-5.16;Fig. 2).There was substantial heterogeneity between studies (I 2 : 61%).A subgroup analysis by the specific type of trapping method used did not explain this heterogeneity.

Fed versus unfed
Analysis from two studies showed that molecular xenomonitoring prevalence wasapproximately 3.5 times higher for mosquitoes collected using fed traps than for mosquitoes collected using unfed traps (prevalence ratio: 3.53; 95% CI: 1.52-8.24;Fig. 3). 26,29Note that 92.7% of the weight of the meta-analysis came  Systematic reviews from a single study 26 that was considered at high risk of bias.A sensitivity analysis excluding this study suggested there was no difference between the two collection methods.

Gravid versus unfed
Only one site provided evidence from sufficiently large effective sample sizes to contribute to the meta-analysis. 27his reported a higher molecular xenomonitoring prevalence for mosquitoes collected using unfed collection methods than for mosquitoes collected using gravid collection methods; however, this finding was not statistically significant (prevalence ratio: 0.20; 95% CI: 0.01-3.40).

Comparison 2: sampling intensity
None of the included studies provided a comparison of different longitudinal collection intensities (for example, nightly collections versus monthly collections).One study compared molecular xenomonitoring prevalence based on different densities of trapping locations (300 versus 150 versus 75 locations) on W. bancrofti detection rates. 33The study found that sampling mosquitoes from 300 locations did not lead to higher molecular xenomonitoring prevalence than when sampling the same number of mosquitoes from 75 sampling locations (prevalence ratio: 0.90; 95% CI: 0.54-1.51).We did not observe any difference in molecular xenomonitoring prevalence between any of the three sampling strategies.

Anopheles versus Culex
9]47 In almost all included studies, the numbers of Culex mosquitoes collected far outweighed the number of Anopheles mosquitoes.In areas where the primary vector is Anopheles, molecular xenomonitoring prevalence was approximately seven times higher for Anopheles mosquitoes than for Culex mosquitoes (prevalence ratio: 6.91; 95% CI: 1.73-27.52;Fig. 4).In areas of Culex-transmitted lymphatic filariasis, the molecular xenomonitoring prevalence was also higher for Anopheles mosquitoes, although the CI for this estimate was wide (prevalence ratio: 2.68; 95% CI: 0.08-94.93).In Anopheles areas, there was substantial heterogeneity between studies (I 2 : 73%) which was not explained by subgroup analyses.The high heterogeneity was due to several studies showing very large differences between mosquito genera.One study in Indonesia reported a much higher prevalence of B. timori DNA in Anopheles mosquitoes than Culex mosquitoes, resulting in a prevalence ratio of 172.7. 37Three additional studies also provided study areas with a prevalence ratio of 20 or greater. 30,31,38

Aedes versus Culex
Two studies from American Samoa and Samoa, comprising 11 study sites, provided comparisons for a metaanalysis of molecular xenomonitoring prevalence between Aedes and Culex mosquitoes. 40,45There was no difference between the two genera (prevalence ratio: 1.07; 95% CI: 0.52-2.19).

Discussion
Our findings suggest that mosquito collection methods can have an important impact on molecular xenomonitoring prevalence, although precise estimates of the impact were difficult to obtain.Multiple studies conducted in Anopheles-transmitted lymphatic filariasis areas reported substantially higher a Downgraded for imprecision: the 95% CIs include both no difference and a large difference.b Downgraded for inconsistency: there was substantial heterogeneity that could not be explained by subgroup analyses.c Downgraded for indirectness: all studies were conducted in areas where the primary filariasis vectors belonged to the Anopheles gambiae species complex; the findings may not apply to other transmission areas.d Downgraded for indirectness: over 92% of the weight of the meta-analysis came from a single study; the findings may not apply to other transmission areas.e Downgraded for risk of bias: the sampling methods for the main study contributing to the analysis were judged to have a high risk bias as mosquitoes collected using fed traps were screened in smaller pool sizes than those collected by gravid traps; this may favour higher molecular xenomonitoring prevalence for mosquitoes from fed collection methods.f Downgraded twice for serious imprecision: the 95% CIs include large differences in favour of each type of collection method.g Downgraded for indirectness: a single study contributed to the meta-analysis; the findings may therefore not apply to other transmission areas.h Downgraded for imprecision: although the 95% CIs are extremely wide and include both a small difference and a very large difference.i Downgraded for indirectness: studies were conducted in American Samoa and Samoa; the findings may not apply to other transmission areas.Note: We used the Grading of Recommendations Assessment, Development and Evaluation approach for grading the quality of the evidence. 22ystematic reviews molecular xenomonitoring prevalence when targeting bloodfed mosquitoes than gravid mosquitoes.However, this effect was not consistent between studies and we were unable to determine the reasons for this heterogeneity.The lack of studies from areas where the primary vector is Culex is an important gap.Gravid traps are an efficient tool for collecting Culex mosquitoes, and have been used as the sole collection method for molecular xenomonitoring by elimination programmes in Bangladesh and Sri Lanka. 2,33ur meta-analysis showed a large difference in molecular xenomonitoring prevalence between fed and unfed collection methods.However, most of the weight of this analysis was contributed by a single study in an area of Cx. quin-quefasciatus-transmitted lymphatic filariasis, 26 and the applicability of this result to other transmission settings is limited.While it is logical that molecular xenomonitoring prevalence would be higher when targeting recently bloodfed mosquitoes, there remains uncertainty from the available data that this effect will always be observed.Strong evidence was reported by one high quality study in Sri Lanka that a reduced number of sampling sites per evaluation unit, from 300 to 75, did not lead to reduced detection of W. bancrofti DNA in Cx. quinquefasciatus.This finding, observed in two post-transmission assessment survey communities, supports the feasibility of molecular xenomonitoring, although evidence from other areas will be required to determine whether this approach is applicable to other transmission zones.

Fig. 3. Effect of mosquito collection method, fed versus unfed, on molecular xenomonitoring prevalence
Lymphatic filariasis programmes looking for the most sensitive approach for detecting Anopheles-transmitted lymphatic filariasis may wish to consider using a sampling strategy that preferentially targets Anopheles mosquitoes, since these methods are likely to collect a higher proportion of parasite-positive mosquitoes.However, this advantage will need to be balanced against the convenience of other collection methods such as gravid traps (which are an efficient method for collecting large numbers of Culex mosquitoes). 9istorically, the collection of bloodfed Anopheles mosquitoes has depended on indoor collections using aspirators or pyrethrum spray -an approach that is labour-intensive and typically results in modest collection numbers. 2 Given the significantly higher molecular xenomonitoring prevalence for Anopheles mosquitoes, programmatic use of molecular xenomonitoring for the detection of ongoing cases of lymphatic filariasis in extremely low prevalence areas may depend upon the development of new tools that are efficient at collecting bloodfed or gravid Anopheles mosquitoes.
We assume that the primary explanation for variation in sensitivity between studies is the differences in the sampling strategy.However, primer and probe design, as well as the equipment used, can affect PCR results, and direct comparisons between molecular xenomonitoring methods have shown variation in sensitivity. 48,49here are narrative reviews on the status of molecular xenomonitoring.For example, one review 50 highlighted the need for systematic methods and new WHO guidelines to be developed to supplement post-validation surveil-lance.Another review 51 proposed that molecular xenomonitoring has enormous potential for the surveillance of vector-borne diseases, with the capacity for it to replace (rather than supplement) current human surveillance strategies.However, they identified several key barriers that must be overcome, including the development of protocols that account for heterogeneity in pathogen infection rates both within the mosquito and the human population.
Our review provides evidence supporting the development of standardized molecular xenomonitoring sampling protocols that specifically consider mosquito collection methods and genus.However, the certainty of evidence for every comparison is very low due to the inherent limitations of observational data, and specific concerns regarding the comparisons drawn from the available literature.

Fig. 1 .
Fig. 1.Flowchart showing the selection of studies included in the systematic review on the impact of mosquito sampling strategies on molecular xenomonitoring prevalence for filariasis

Table 2 . Methodological quality assessment of studies included in the systematic review on the impact of mosquito sampling strategies on molecular xenomonitoring prevalence for filariasis
Note: Some articles are included in more than one comparison and hence appear twice in the table.Bull World Health Organ 2024;102:204-215| doi: http://dx.doi.org/10.2471/BLT.23.290424

Table 3 . The relative effect of different mosquito sampling strategies on molecular xenomonitoring prevalence and evaluations of the certainty of the evidence
CI: confidence interval.